Cell-to-cell variability in troponin I phosphorylation in a porcine model of pacing-induced heart failure

We tested the hypothesis that myocardial contractile protein phosphorylation and the Ca2+ sensitivity of force production are dysregulated in a porcine model of pacing-induced heart failure (HF). The level of protein kinase A (PKA)-dependent cardiac troponin I (TnI) phosphorylation was lower in the myocardium surrounding the pacing electrode (pacing site) of the failing left ventricle (LV) than in the controls. Immunohistochemical assays of the LV pacing site pointed to isolated clusters of cardiomyocytes exhibiting a reduced level of phosphorylated TnI. Flow cytometry on isolated and permeabilized cardiomyocytes revealed a significantly larger cell-to-cell variation in the level of TnI phosphorylation of the LV pacing site than in the opposite region in HF or in either region in the controls: the interquartile range (IQR) on the distribution histogram of relative TnI phosphorylation was wider at the pacing site (IQR = 0.53) than that at the remote site of HF (IQR = 0.42; P = 0.0047) or that of the free wall of the control animals (IQR = 0.36; P = 0.0093). Additionally, the Ca2+ sensitivities of isometric force production were higher and appeared to be more variable in single permeabilized cardiomyocytes from the HF pacing site than in the healthy myocardium. In conclusion, the level of PKA-dependent TnI phosphorylation and the Ca2+ sensitivity of force production exhibited a high cell-to-cell variability at the LV pacing site, possibly explaining the abnormalities of the regional myocardial contractile function in a porcine model of pacing-induced HF

Effects of neonatal capsaicin desensitization on sensory TRPV1 expression.

<p>TRPV1 expression was examined in the dorsal root ganglia of control and capsaicin desensitized (neonatal capsaicin treatment) adult rats. Tissue sections were probed with antibodies specific to TRPV1 (red) and neurofilament (green). TRPV1 expression was present in a subset of sensory neurons in control rats, which was missing in desensitized rats within the dorsal root ganglia. However, TRPV1 immunoreactivity was also found in non-neuronal cell types, located at the outer regions of ganglia (regions occupied by sensory neurons are indicated by the freehand drawing). Images are representative of at least 10 sections.</p

Functional effects of acute capsaicin desensitization on vascular TRPV1.

<p>The short-term effects of capsaicin were tested here. Arterioles were isolated from control rats and were treated with capsaicin (1 µM, First treatment). Capsaicin was washed away after the 20 min long treatment and the arterioles were incubated in the physiological buffer alone for 40 min (regeneration). Then capsaicin effects we re-tested (Second treatment) to estimate the level of tachyphylaxis (n = 6 arteries were tested, symbols represent the mean ± SEM of time-matched arteriolar diameter). Acute desensitization to capsaicin was apparent upon the first treatment (decrease and then increase in arteriolar diameter in the continuous presence of capsaicin, maximal decrease was 70±3% at 95 s after capsaicin application, Panel A). The magnitude of the response was decreased upon the second capsaicin treatment on the same arterioles (tachyphylaxis, decreased response to repeated capsaicin stimuli, maximal decrease was 27±9% at 125 s after capsaicin application, Panel A). This acute capsaicin desensitization (20 min treatment) did not affect norepinephrine mediated constrictions (symbols represent the mean ± SEM of n = 6 determinations, Panel B) or acetylcholine mediated dilations (symbols represent the mean ± SEM, n = 6, Panel C).</p

Effects of neonatal capsaicin desensitization on sensory functions.

<p>Rats were treated with saline (Control) or with capsaicin (Desensitized) at 14 days of life. Sensory functions were measured 10 weeks after capsaicin treatment. (A) Eye wiping (a measure of capsaicin evoked sensory irritation) was reduced in desensitized rats (n = 10 in both groups, p<0.001). (B) Plasma extravasation was similarly reduced (Evans blue accumulation, a measure of capsaicin mediated neurogenic inflammation) (n = 9 for Control and n = 5 for Desensitized, p = 0.03).</p

TRPV1 immunoreactivity in vascular smooth muscle cells.

<p>Rat dorsal root ganglia sections were co-stained by anti TRPV1 (red) and anti vascular smooth muscle actin (SMA, green) antibodies. Some of the TRPV1 positive cells were co-stained by the antibody specific to vascular smooth muscle actin, suggesting TRPV1 expression in this cell type. Moreover, vascular TRPV1 expression was not affected by neonatal capsaicin treatment (note similar TRPV1 staining in SMA positive regions of control and desensitized rats). Images are representative of at least 6 sections.</p

Effects of neonatal capsaicin desensitization on vascular TRPV1 expression.

<p>Skeletal muscle (gracilis muscle of the rat) tissue sections were stained with anti-TRPV1 (red) and anti-vascular smooth muscle actin (SMA, green) antibodies. There appeared a complete overlap of the staining patterns (merged images). Arteriolar TRPV1 expression was not affected by neonatal capsaicin desensitization, similarly to the vascular TRPV1 in the dorsal root ganglia. Images are representative of at least 10 sections.</p

Functional effects of neonatal capsaicin desensitization on arteriolar TRPV1.

<p>Effects of neonatal capsaicin desensitization were characterized on the functional properties of isolated, cannulated skeletal muscle arterioles. Endothelial responses were tested by the application of acetylcholine (Panel A, values normalized to the maximal acetylcholine mediated dilations, symbols represent the mean ± SEM, n = 4 for the control and n = 4 for the desensitized). Smooth muscle functions were assessed by norepinephrine (Panel B, symbols represent the mean ± SEM of n = 4 determinations in both cases). The spontaneous myogenic tone (Panel C, symbols represent the mean ± SEM of n = 4 determinations for all groups) was tested by the contractile response developing in response to increasing intraluminal pressure (from 20 mmHg to 120 mmHg in 20 mmHg increments). Finally, capsaicin mediated responses were also tested by the cumulative application of the drug. Capsaicin evoked a dose-dependent constriction, supporting a physiological role for this receptor in vascular smooth muscle cells, which was not affected by the desensitization protocol (Panel D, symbols represent the mean ± SEM of n = 4 determinations for both groups).</p